Ethylene Glycol-supplemented Calcium-free Media Improve Zona Penetration of Vitrified Rat Oocytes by Sperm Cells
Ethylene Glycol-supplemented Calcium-free Media Improve Zona Penetration of Vitrified Rat Oocytes by Sperm Cells
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DOI：
10.1262/jrd.09-107h
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发表时间：
2010-02-01
影响因子：
1.8
通讯作者：
Kashiwazaki, Naomi
中科院分区：
文献类型：
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作者：
Fujiwara, Katsuyoshi;Sano, Daisuke;Kashiwazaki, Naomi
冷冻保存成熟卵母细胞是一种有用的技术，因为卵母细胞可以在加热后用于一些辅助生殖技术。尽管与小鼠一样，大鼠已被用于包括生殖技术在内的各种研究领域，但有关大鼠卵母细胞冷冻保存的信息有限。本研究的目的是改进成熟大鼠卵母细胞的玻璃化方法。为了确定最佳的平衡时间，卵母细胞分别在7.5%乙二醇(EG) + 7.5%二甲基亚砜(DMSO) + 20%胎牛血清（FCS）中在24℃下平衡1,4,7或10 min，然后在24℃下，15%乙二醇+ 15% DMSO + 0.5 M蔗糖+ 20% FCS 1 min，然后在Cryotops上投入液氮。卵母细胞在平衡培养基中暴露4 min后，人工激活后的存活率和卵裂率高于暴露1、7和10 min的卵母细胞。活化后的卵母细胞存活率为98.3%，卵裂率为78.4%。然而，大多数玻璃化/加热的卵母细胞（6/168,3.6%）在体外受精后精子不能穿透卵泡周围间隙，并且在卵母细胞中观察到皮质颗粒胞吐（CGE）。因此，研究了玻璃化介质中钙和冷冻保护剂对CGE的抑制作用。在大多数在无钙培养基中玻璃化的卵母细胞中，CGE被强烈抑制，不依赖于冷冻保护剂。卵母细胞在添加了卵细胞的无钙培养基中玻璃化，加热后存活率高（79.4%）。人工激活后，卵母细胞的卵裂率和囊胚形成率也很高（分别为72.8%和23.1%）。体外受精后，添加无钙培养基后，玻璃化/加热的卵母细胞带渗透率显著提高（111/155,63.9%）。因此，我们的数据表明，补充蛋氨酸的无钙培养基可以改善精子细胞对玻璃化大鼠卵母细胞的渗透。
Cryopreservation of matured oocytes is a useful technique because the oocytes can be used for some assisted reproductive technologies after warming. Even though rats, like mice, have been used in various research fields including reproductive technology, information about cryopreservation of rat oocytes is limited. The objective of the present study was to improve the vitrification protocol for matured rat oocytes. To determine the optimal equilibration time, oocytes were equilibrated in 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% fetal calf serum (FCS) for 1, 4, 7 or 10 min at 24 C and then 15%, EG + 15%, DMSO + 0.5 M sucrose + 20%, FCS for 1 min at 24 C before being plunged into liquid nitrogen on Cryotops. Oocytes exposed to equilibration medium for 4 min showed higher survival and cleavage rates after artificial activation than those of oocytes exposed for 1, 7 or 10 min. The survival and cleavage rates of vitrified oocytes after activation were 98.3 and 78.4%, respectively. However, the perivitelline spaces of most of the vitrified /warmed oocytes (6/168, 3.6%) could not be penetrated by sperm after in vitro fertilization, and cortical granule exocytosis (CGE),vas observed in the oocytes. Therefore, the inhibitory effect of calcium and cryoprotectants in vitrification medium on CGE was examined. In most of the oocytes vitrified in calcium-free media, CGE was strongly suppressed independent of cryoprotectants. Oocytes vitrified in EG-supplemented calcium-free media showed high survival rates after warming (79.4%). After artificial activation, the cleavage and blastocyst formation rates of the oocytes were also high (72.8 and 23.1%, respectively). The zona penetration rate of vitrified/ warmed oocytes was dramatically improved by using EG-supplemented calcium-free media after in vitro fertilization (111/155, 63.9%). Thus, our data suggest that EG-supplemented calcium-free media improve zona penetration of vitrified rat oocytes by sperm cells.